The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.

FIG. 1

Schematic representation of asynchronous and synchronous cultures. The genotype shows that cells in G₁ rely on NHEJ (haploids) or interhomologue recombination (diploids) for recombinational DNA repair. Cells in G₂ can also undergo sister chromatid recombination (haploids and diploids). The petal-shaped figures represent chromosome arms; the gray dots represent the centromeres.

FIG. 2

Radiation sensitivity of haploid mre11Δ and hdf1Δ strains. Asynchronous cultures and cells synchronized with α-factor (G₁ synchronous) or carbendazim (G₂ synchronous) were irradiated at a dose of 150 Gy as described in Materials and Methods. Cell survival was scored for 5 days following irradiation. Values plotted represent the average of triplicate platings from at least three experiments. Error bars represent standard deviations. Haploid strains were JPY70 (MRE11), JPY69 (mre11Δ), JPY181 (hdf1Δ), and JPY254 (mre11Δ hdf1Δ).

FIG. 3

Sister chromatid recombination assay. Integrative transformation of MFp102 (11) results in the replacement of the TRP1 locus with the SCR substrate. Striped bars indicate the location of his3 fragments (his3-Δ5′ and his3-Δ3′) in a head-to-tail arrangement along the sister chromatids (solid horizontal lines). The arrowhead and shaded box represent regions of HIS3 deleted in the opposite allele. A functional HIS3 gene can be generated only by an unequal sister chromatid recombination event as indicated by the diagonal line. An intrachromatid recombination event will produce a HIS3 circle (not shown).

FIG. 4

Radiation sensitivity of diploid mre11Δ and mre11Δ hdf1Δ strains. Methods of synchronization and irradiation are as described in the legend to Fig. 3 and in Materials and Methods. IR dose = 150 Gy. Values plotted represent the averages of triplicate platings from at least three experiments. Error bars represent standard deviations. Diploid strains were JPY146 (MRE11), JPY145 (mre11Δ), and JPY260 (mre11Δ hdf1Δ).

FIG. 5

Radiation sensitivity of synchronous mre11-3 cultures. Methods of synchronization and irradiation are as described in the legend to Fig. 3 and Materials and Methods. IR dose = 150 Gy. Haploid mre11Δ strain JPY69 was transformed with an ADH1 promoter-driven MRE11 or mre11-3 expression construct (DB-MRE11-TRP or DB-mre11-3-TRP) or an empty vector (DB-P-TRP) (7). Asynchronous, G₁-synchronous, and G₂-synchronous cultures of JPY69 transformants were plated onto SC-Trp media and scored for cell survival for 5 days following irradiation. Values plotted represent the averages of triplicate platings. Error bars represent standard deviations. The standard deviation for the asynchronous MRE11 culture (1.2%) is not visible at the scale of this plot.